The ability of some bacteria to survive antibiotic treatment is a serious problem for the treatment of infectious diseases. This is especially true fo the pathogen Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB). Global TB mortality rates are increasing, despite the aggressive and lengthy antibiotic chemotherapy used to treat the disease. One of the reasons for failed treatment is that even in drug susceptible disease a considerable population of bacteria can survive antibiotic therapy. I intend to study this phenomenon of phenotypic tolerance, often referred to as persistence, in mycobacteria. Phenotypic tolerance is poorly understood and difficult to study since it requires looking at individual cells rather than populations. Here, I propose a novel method based on fluorescent biosensors and time-lapse microscopy to test my hypothesis that bacterial physiological state predetermines antibiotic susceptibility on a single-cell level and that this stte can fluctuate in response to endogenous and exogenous stimuli. The results will give insight into the mechanisms underlying phenotypic tolerance in mycobacteria, leading to future work that will focus on developing new strategies for treating TB infections more rapidly.